1. Tryptophan synthase We have continued work on the high resolution structure of the bifunctional enzyme complex tryptophan synthase. This complex is a paradigm for the study of intersubunit communication and allosteric activity. We have examined the mutant betaK87T which exists as the external aldimine combined with serine and with tryptophan. A series of conformational differences have been observed that provide a basis for understanding the mechanism of action and the communication between the two active sites. 2. Deoxyhypusine synthase The crystal structure of the enzyme Deoxyhypusine synthase has been determined at 2.2A resolution. The enzyme is a homotetramer consisting of two pairs of closely linked subunits with the active sites shared between the subunits. The active site is located in a deep cavity in which the spermidine is presumably bound. The nicotinamide ring is oriented in a manner consistent with the established stereospecificity of the hydride reaction. One interesting feature of the structure is that there is an N- terminal helix from a third subunit that forms a ball and chain arrangement which is located so as to block access to the active site. Attempts are now being made to crystallize the enzyme in the presence of its substrate, initiation factor 5A. 3. Serotonin N-acetyltransferase This enzyme is penultimate in the conversion of serotonin to melatonin, the nocturnal neurohormone produced in the pineal gland. The structure of ovine NAT has been determined at 2.5A resolution. The protein consists of a central beta sheet flanked by alpha-helices. Two parallel strands in the center of the beta-sheet are pulled apart at their C-terminal ends, and form a "V"-shaped binding site for the cofactor, acetyl coenzyme A. This permits the identification of two histidine residues that could act as catalytic bases; furthermore, site directed mutagenesis results indicate that conservative mutation of either of these residues renders the protein inactive. On the basis of this structure a detailed catalytic mechanism has been proposed which is now in the process of being tested through mutagenic and kinetic analyses. 4. Tn7 Transposon The Tn7 transposon is a mobile DNA element that moves from DNA site to DNA site using a complex array of self-encoded proteins. The transposon carries genes encoding for enzymes that confer resistance to trimethoprim and the aminoglycosides streptomycin and spectinomycin, and exemplifies one method by which multi-drug resistance can be transferred between organisms. One of the proteins which forms part of the transposase, TnsA, has recently been crystallized in the presence of its cofactor, Mg++, and native data have been collected to 2.4A. Structure determination is in progress.